The art has long appreciated that one time use of enzymes to conduct a desired enzymatic reaction may involve such an inordinate enzyme cost as to make conduct of the reaction prohibitively expensive.
In particular, it is known that glucose can be isomerized enzymatically to a much sweeter, about 50--50 mixture of glucose and fructose, but the enzyme expense is great and the isomerization procedure must be tailored to match the characteristics of the enzyme. Since a reusable enzyme offers hope for a low cost process, the possibility for multiple reuse of enzymes has not escaped the attention of the art. Numberous proposals have been made to stabilize and/or alternatively, to immobilize both cellular and cell free glucose isomerase enzymes.
Since enzyme reuse requires recovery of the enzyme from the reaction mixture, soluble enzymes are insolubilized and attached to a matrix of some sort. In the instance of glucose isomerase, an intracellular enzyme, the enzyme is already bonded to or entrapped inside the microbial cell, but leaching out of the enzyme and/or disintegration of the cell must be avoided. Also, microbial cells are quite tiny. Larger particles would be more desirable.
A related point of some significance to isomerization of glucose is the desirability of being certain that reagents employed to stabilize or encapsulate the glucose isomerase containing cells will not release materials that constitute a detriment to the final glucose/fructose syrup. Some of the immobilization techniques suggested to the art may never be employed in commercial practice because reagents and reaction products have not been accepted as non-toxic. In this regard, reaction of microbial cells with glutaraldehyde offers an advantage since glutaraldehyde reaction products are generally recognized as ingestible (certainly in the miniscule quantities that might appear in the syrup product).
In this connection, reference is made to U.S. Pat. No. 3,779,869 as being directed to stabilization of glucose isomerase containing bacterial cells by reaction with glutaraldehyde. Other suggestions have been made to the art to cross-link various enzymes by a reaction with glutaraldehyde.
Glutaraldehyde is known to react with (amino) nitrogen containing materials, even enzymes. However, whole cells of glucose isomerase microorganisms are simply not reactive enough with glutaraldehyde to cross-link into multi-cell particles. Suggestions for cross-linking, insolubilization and immobilization of enzymes have involved inclusion of an extraneous reactant with glutaraldehyde ingredient (such as for example albumin) for covalent bonding of enzymes thereto. Reference is made to British Pat. No. 1,257,263 for detailed description of such (and other like) expedients. Unfortunately, addition of extraneous reactant, in the needed quantities significantly dilutes the glucose isomerase, lowering the unit activity of the product proportionate to the dilution. Higher unit activity immobilized products can, of course, be prepared if the glucose isomerase is removed from the cell and purified prior to immobilization. However, the processing costs and activity loss incident to processing sharply increase the expense of any high activity product.
The situation is particularly troublesome in the instance of glucose isomerase. Isomerization of glucose on an industrial scale requires very large quantities of a relatively low cost enzyme product with the highest possible unit activity. Stabilized cells would seem best suited to this requirement, but providing for use, recovery and reuse of individual cells on an industrial scale is an engineering nightmare. If a significant reuse factor, e.g. five or more times, can be attained, the art is willing to accept the somewhat lowered unit activity inherent in a particulate product (far larger than microorganism cells).
It has now been discovered that a controlled particle size glucose isomerase product can be prepared without diluting the enzyme content by inclusion of an extraneous immobilization reactant. It has been ascertained that the cells themselves contain more than enough nitrogenous (and perhaps other) constituents reactive with glutaraldehyde to create a gel product. Such constituents must first be liberated from the microorganism cells, but when liberated will serve the purpose without need for intermediate purification.
Liberation of nitrogenous constituents such as for example proteins and nucleic acids can be effected by mechanical action or autolysis. The liberation need not be complete. Fracturing as little as 25% of the cells can release sufficient reactants to allow creation of a gel product.